The major objective of this application is to gain mechanistic insights into the roles of Tet-induced oxidation products of 5-methyl-2'-deoxycytidine (5-mdC) in epigenetic regulation in mammals. Methylation at the C5 position of cytosine at CpG dinucleotide sites constitutes a major mechanism of epigenetic regulation in mammals. It was not clear whether 5-mdC in the mammalian genome can be converted to its unmethylated counterpart through a process that is independent of DNA replication (a.k.a. active cytosine demethylation). Recent discovery of the functions of ten-eleven translocation (Tet) family of enzymes offered important new insights into active cytosine demethylation in mammals. Tet enzymes can oxidize 5-mdC in DNA to yield 5- hydroxymethyl-2'-deoxycytidine (5-HmdC), 5-formyl-2'-deoxycytidine (5-FodC) and 5-carboxyl-2'-deoxycytidine (5-CadC). In addition, 5-FodC and 5-CadC can be efficiently cleaved from DNA by thymine DNA glycosylase, and subsequent action by the base excision repair machinery can result in the ultimate replacement of 5-mdC with an unmethylated dC. In this R21 application, we propose experiments to explore the novel mechanisms of Tet-mediated oxidation products in epigenetic regulation and the proposed research is organized according to the following two specific aims: (1) To exploit the cellular roles of de novo DNA cytosine methyltransferases, DNMT3a and DNMT3b, in the direct conversions of 5-HmdC, 5-FodC and 5-CadC to unmethylated dC. We will assess the functions of DNMT3a and DNMT3b in the transformations of 5-HmdC, 5-FodC and 5-CadC to unmethylated dC in human cells and how this process is modulated by histone epigenetic marks. (2) To examine the occurrence, biosynthesis and transcriptional perturbation of potential glucose-conjugated derivatives of 5-HmdC in human cells. Based on our newly developed method for the quantification of base J in trypanosome DNA, we will examine the formation of the analogous glycosylated 5-HmdC in human cells, identify the potential enzyme(s) involved in this conversion, and assess the impact of the glycosylated 5-HmdC on transcription in human cells. The outcome of the proposed research will yield important new knowledge for understanding the role of Tet-mediated oxidation products of 5-mdC in epigenetic regulation in mammals. Exposure to many environmental agents is known to stimulate the generation of reactive oxygen species, which could also result in the inadvertent oxidation of 5-mdC to 5-HmdC, 5-FodC and 5-CadC. Thus, the proposed research may also provide new knowledge for understanding how environmental exposure perturbs epigenetic mechanisms of gene regulation.